ABSTRACT The need for an effective therapy for pancreatic ductal adenocarcinoma (PDAC) has prompted us to develop a novel combinatorial immunotherapeutic strategy for this very aggressive malignancy. In the design of our strategy we have been guided by the realization that to be effective, a therapy has to eradicate both differentiated PDAC cells and PDAC cancer initiating cells (CICs). According to the cancer stem cell theory, CICs play a major role in disease recurrence and metastatic spread, the major causes of patient morbidity and mortality. Furthermore, immunotherapy has to counteract the multiple escape mechanisms utilized by PDAC cells to avoid immune destruction. Therefore, i) to eliminate both differentiated PDAC cells and PDAC CICs, we have selected as a target B7-H3, since this tumor antigen (TA) can mediate the immune destruction of both types of targets. In addition, B7-H3 is up-regulated on tumor associated fibroblasts and stroma in the tumor microenvironment. Therefore, B7-H3 immunotargeting inhibits neoangiogenesis and contributes to the elimination of PDAC cells, even those with B7-H3 loss or down regulation. Lastly B7-H3 has a restricted distribution in normal tissues. Therefore, B7-H3 immunotargeting is not expected to cause side effects because of the targeting of normal tissues. ii) To counteract the escape mechanisms caused by abnormalities in HLA class I antigen processing machinery in PDAC cells, we have selected as effectors NK cells which do not require HLA class I antigen expression for recognition of target cells. To activate and expand the NK cell effector population at the level of the immune synapse, we have added IL-15 to the conventional bispecific NK Cell immune engagers (BiKEs) platform and used it to crosslink the scFv fragments derived from the B7-H3-specific mAb 376.96 to a highly modified camelid CD16 (FC?RIII)-specific scFv fragment. The latter binds to NK cells, while the former to PDAC cells. These novel hybrid molecules which are referred to as IL-15 TriKEs enhance the ability of NK cells to kill tumor cells with limited - if any - damage to normal cells. iii) To restore the susceptibility of PDAC cells to immune lysis we combine IL-15 TriKEs with the small molecule sonidegib, an inhibitor of the sonic hedgehog homolog pathway, since the activation of this pathway up-regulates the level of anti-apoptotic molecules. iv) To enhance the anti-tumor activity of NK cells we disrupt the PD-1/PD-L1 axis with an anti-PD-L1 mAb. We will test the hypothesis that NK cells in combination with IL-15 TriKEs, sonidegib and anti-PD-L1 mAb are effective in eradicating both differentiated PDACC cells and PDACC CICs, both in vitro and in vivo. As a result, PDAC disease recurrence and metastatic spread will be suppressed. Because of the limited budget the proposed studies will be performed only with PDAC cell lines. In future studies the potential clinical significance of the results obtained with cell lines will be assessed with patient derived xenografts. This information will then be translated to a clinical setting in a Phase I clinical trial.